Phase I study of sapanisertib (CB‐228/TAK‐228/MLN0128) in combination with ziv‐aflibercept in patients with advanced solid tumors

Abstract Background Sapanisertib is a potent ATP‐competitive, dual inhibitor of mTORC1/2. Ziv‐aflibercept is a recombinant fusion protein comprising human VEGF receptor extracellular domains fused to human immunoglobulin G1. HIF‐1α inhibition in combination with anti‐angiogenic therapy is a promising anti‐tumor strategy. This Phase 1 dose‐escalation/expansion study assessed safety/ tolerability of sapanisertib in combination with ziv‐aflibercept in advanced solid tumors. Methods Fifty‐five patients with heavily pre‐treated advanced metastatic solid tumors resistant or refractory to standard treatment received treatment on a range of dose levels. Results Fifty‐five patients were enrolled and treated across a range of dose levels. Forty were female (73%), median age was 62 (range: 21–79), and ECOG PS was 0 (9, 16%) or 1 (46, 84%). Most common tumor types included ovarian (8), colorectal (8), sarcoma (8), breast (3), cervical (4), and endometrial (4). Median number of prior lines of therapy was 4 (range 2–11). Sapanisertib 4 mg orally 3 days on and 4 days off plus 3 mg/kg ziv‐aflibercept IV every 2 weeks on a 28‐day cycle was defined as the maximum tolerated dose. Most frequent treatment‐related grade ≥2 adverse events included hypertension, fatigue, anorexia, hypertriglyceridemia, diarrhea, nausea, mucositis, and serum lipase increase. There were no grade 5 events. In patients with evaluable disease (n = 50), 37 patients (74%) achieved stable disease (SD) as best response, two patients (4%) achieved a confirmed partial response (PR); disease control rate (DCR) (CR + SD + PR) was 78%. Conclusion The combination of sapanisertib and ziv‐aflibercept was generally tolerable and demonstrated anti‐tumor activity in heavily pre‐treated patients with advanced malignancies.


| INTRODUCTION
The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is one of the most frequently deregulated pathways in human cancer and a key regulator of cellular proliferation, growth, and survival. 1][6][7][8][9][10] Consequently, targeting this pathway has been the focus of oncology trials for many decades.
mTOR is a serine-threonine kinase and a key intracellular point of convergence for several pathways in human cancer and thus represents an important therapeutic target.mTOR exists as two complexes, one with raptor, mTOR complex 1 (mTORC1) which is rapamycinsensitive, and the other with rictor, which is typically rapamycin insensitive (mTORC2). 11mTOR complex 1 (mTORC1) phosphorylates 4EPB1 and p70S6 kinase and results in translation of proteins involved in cell cycle progression, while rapamycin-insensitive mTORC2 has been shown to directly phosphorylate and activate AKT at serine 473. 12Inhibition of mTORC1 inhibits the negative feedback loop between S6 kinase and insulin receptor substrate which results in an increase in PI3K and AKT activity which may limit the activity of rapamycin and impair rapalog efficacy. 13,146][17] Sapanisertib (formerly TAK-228 or MLN0128) is a potent, selective, oral dual inhibitor of mTORC1 and TORC2, developed to address incomplete inhibition of mTOR by rapalogs.9][20][21] In addition, recently published early phase clinical trials have demonstrated promising anti-tumor activity and manageable safety profile of single agent sapanisertib in endometrial and renal cell carcinoma. 22ngiogenesis has been implicated in tumor development and metastasis, 23 and is partly mediated by vascular endothelial growth factor (VEGF). 24The anti-angiogenic properties of mTOR inhibitors have been well documented in both in vitro and in vivo models. 25,26There are pre-clinical data to suggest that hypoxia-inducible factor 1 α (HIF-1α) is modulated by mTORC1, 27 and that mTORC1 drives HIF-1α and VEGF-A signaling via multiple signaling mechanisms involving 4E-BP1, S6K1, and STAT3. 27Furthermore, STAT3 has been shown to be directly phosphorylated by mTORC1 on Ser727 during hypoxia, promoting HIF-1α mRNA transcription.Increased levels of HIF-1α have been associated with increased expression of VEGF, aggressive tumor growth, and poor patient prognosis. 13his phenomenon has been observed as a mechanism of resistance in tumors treated with anti-VEGF therapy. 28TOR pathway inhibition together with VEGFR pathway inhibition has shown synergism in renal cell carcinoma using pre-clinical models. 29HIF-1α inhibition in combination with anti-angiogenic therapy may further strengthen the capabilities of angiogenesis inhibitors 30 and is a promising strategy for targeting tumor resistance. 28,31iv-aflibercept is a recombinant fusion protein consisting of human VEGF receptor extracellular domains fused to the Fc portion of human immunoglobulin G1 (IgG1) and contains portions of the extracellular domains of 2 different VEGFRs: VEGFR1 (also known as Flt-1) and VEGFR2 (also known as KDR or Flk-1).mTOR pathway inhibitors, such as sapanisertib, inhibit the activity of several angiogenic factors, including HIF-1α, which result in decreased VEGF and decreased angiogenic activity. 31oreover, a phase III study of everolimus in pancreatic neuroendocrine tumors demonstrated that mTOR inhibition may reduce circulating levels of sVEGFR1, PlGF, and bFGF. 32ltogether, these data suggested that sapanisertib would be a suitable candidate for combination therapy with ziv-aflibercept and we hypothesized that combination therapy could enhance anti-tumor activity of sapanisertib and target tumor resistance.Here, we report the preliminary safety, tolerability, and efficacy from the doseescalation study of sapanisertib in combination with aflibercept in patients with advanced solid tumors.

| Study design
This was an open-label, single-center Phase I clinical trial that employed a 3 + 3 dose-escalation design and was conducted at The University of Texas M.D. Anderson Cancer Center and supported by NCI-CTEP.The primary endpoint was to evaluate the safety and tolerability of sapanisertib in combination with ziv-aflibercept, to determine maximum tolerated dose (MTD) and dose-limiting toxicities (DLT) of the combination in patients with advanced cancers refractory to standard therapy.Secondary objectives included the evaluation of preliminary anti-tumor efficacy of the combination treatment per response evaluation criteria in solid tumors (RECIST version 1.1) 33 and to evaluate AKT/mTOR signaling and adaptive responses.
One cycle consisted of 4 weeks of treatment (28 days), with sapanisertib taken orally for 3 days on and 4 days off starting on Cycle 1 Day 2 (and starting Day 2 of every cycle).Ziv-aflibercept was given via intravenously (IV) infusion once every 2 weeks (Days 1 and 15 for each cycle).Both study drugs were permitted a +/− 2 day window, though not permitted to be given on the same day.
Trial enrolment was challenging due to need to re-start the dose escalation three times, which included modification to the dosing schedule, and drug formulation changes.There were three original planned dose escalations scheduled (Table S1).These dose levels were subsequently modified, and patients were treated in the dose levels as shown in Table 3.During dose-escalation, patients received one of four sapanisertib doses (3 mg/ 4 mg/ 5 mg / 6 mg) once daily (QD) for 3 days on/4 days off in combination with ziv-aflibercept in one of three doses (2, 3 or 4 mg/kg) given intravenously IV every 2 weeks, in a 28-day cycle (Table 3).Once the MTD was determined, the expansion cohort was opened (Table S2).Tumor measurements were performed every 8 weeks.Data cut-off date was December 15, 2021.There were no planned intra-patient dose escalation, and no patients were enrolled in the next dose level until the toxicity was fully assessed following completion of 1 cycle and at least 3 patients enrolled at the previous dose level.
Per study protocol, DLTs were defined as an adverse events (AE) occurring within the first cycle deemed related to study agents with an attribution of possible, probably, or definite and fulfilling one of the following criteria: grade 4 neutropenia lasting >7 days, febrile neutropenia (defined as absolute neutrophil count [ANC] <1.0 × 10 9 /L and fever ≥38.5°C) or documented grade ≥3 infection with ANC ≤1.0 × 10 9 /L, platelet count <25,000/mm lasting >7 days, and any grade ≥3 non-hematologic toxicity that persists for >7 days, except for the following: nausea/vomiting, diarrhea, and electrolyte imbalances; grade 3 laboratory abnormalities that are asymptomatic and responsive to supportive measures and that are without clinical consequence; grade 3 hyperglycemia or grade 3 diabetes that can be stably controlled; grade 3 laboratory abnormalities that are asymptomatic and responsive to supportive measures and were without clinical consequence; and grade 3 hypertension that resolved within 14 days with medical management.Patients who experienced proteinuria that resolved to <2 g within 14 days were not defined as a DLT.Hypersensitivity/Allergic reactions with expected severity and presentation were not considered a DLT.Patients who withdrew from the study prior to completion of the first cycle of study treatments for reasons other than treatmentrelated AEs were replaced.

| Patients
All patients provided written informed consent for participation.Eligible patients included patients with metastatic or advanced solid tumor resistant or refractory to standard therapy, an Eastern Cooperative Oncology Group (ECOG) performance status 0 to 1 and adequate hematologic, hepatic, and renal function.Patients with known brain metastases were excluded from the study.Patients had to have the ability to swallow oral medications and patients with uncontrolled diabetes mellitus (defined as fasting serum glucose >130 mg/dL despite best medical management or HbA1c >7%) and uncontrolled hypertension (defined as blood pressure >150/95 mmHg, or systolic blood pressure >180 mmHg when diastolic blood pressure <90 mmHg, on at least 2 repeated determinations on separate days within 3 months prior to study enrolment) were excluded.Prior treatment with mTOR inhibitors, TORC1/2 inhibitors, TORC1 inhibitors, or AKT inhibitors was permitted.Patients with history of significant cardiovascular or pulmonary disease, intercurrent uncontrolled illness, malabsorption due to prior gastrointestinal (GI) surgery, and GI disease (eg.patients with enteric stomata were excluded) were excluded.Urine protein screen by dipstick or urine analysis was required; for proteinuria >1+ or urine protein: creatinine ratio >1.0, 24-hour urine protein was recommended to be obtained and level of <2000 mg was required for patient enrollment.Patients were permitted to have evaluable or measurable disease by RECIST v1.1. 33

| Assessments
Data from all patients who received one or more doses of drug were incorporated into the final safety analysis.All patients who receive any amount of the study drug(s) were evaluable for toxicity.The MTD was defined as the highest dose level at which no more than 1 of 6 evaluable patients experienced a DLT.If multiple toxicities were seen, the presence of DLT was based on the most severe toxicity experienced.The responseevaluable population was defined as all patients who had measurable disease at baseline according to RECIST version 1.1, who had received at least 1 dose of any study drug, and who had at least 1 available post-baseline response assessment as per RECIST version 1.1.Response was assessed according to the RECIST v1.1 following every two cycles of treatment.AEs were assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events, (NCI CTCAE version 4.0 until March 31, 2018, and version 5.0 beginning April 1, 2018).Detailed descriptions of predefined DLTs, management of AEs, and safety and efficacy assessments are included in the Protocol.

| Statistical analysis
No formal hypotheses were tested, and analyses were descriptive and exploratory.

| Treatment response
Overall, there were 50 response-evaluable patients, including five patients with non-measurable but evaluable disease.Five patients were not evaluable for response as they came off treatment prior to first re-staging scan.Of the 50 patients evaluable for response, two patients (4%) had confirmed PR, 37 patients (74%) had SD, including four patients with unconfirmed PR, and 11 patients (22%) had PD, including two patients with clinical progression of disease (Table S3).The overall response rate (ORR) was 4%, and disease control rate (DCR; CR + SD + PR) was 78% Renal cell 2 Head and neck SCC 2 (4) Cancer of unknown primary 1 (Figure 1).The most frequent reason for study discontinuation was PD in 64% of patients.PRs were achieved at dose level 1 (n = 1), dose level 2 (n = 1), and dose level 3 (n = 2), and these unconfirmed partial responses were observed in patients with leiomyosarcoma (n = 2), breast cancer (n = 1), and endometrial cancer (n = 1), (Figure 2).The most frequent reason for study discontinuation was progression of disease (PD) in 64% of all patients.

| DLTs and MTD determination
Dose escalation, DLTs, and MTDs are summarized in Table 3. Doses were escalated to modified dose level 6, sapanisertib 6 mg orally 3 days on and 4 days off plus 4 mg/kg ziv-aflibercept IV every 2 weeks on a 28day cycle (Table 3).There were no DLTs reported until this cohort was treated (two DLTs in two separate patients).One patient (1 out of 4) experienced DLT of grade 3 transient ischemic attack (TIA) related to zivaflibercept; the second patient experienced DLT of grade 3 gastritis.Doses were reduced to next dose level (dose level 5), sapanisertib 5 mg orally 3 days on and 4 days off plus 3 mg/kg ziv-aflibercept IV every 2 weeks on a 28day cycle (Table 3), where one patient experienced DLT with grade 3 dyspnea, possibly related to both study drugs.Doses of both study drugs were modified, and the patients were treated in the expansion cohort using sapanisertib 4 mg orally 3 days on and 4 days off plus 3 mg/ kg ziv-aflibercept IV every 2 weeks on a 28-day cycle.No DLTs observed in the expansion cohort.Based on these findings, sapanisertib 4 mg orally 3 days on and 4 days T A B L E 2 Treatment-related adverse events in all patients receiving sapanisertib in combination with ziv-aflibercept.off plus 3 mg/kg ziv-aflibercept IV every 2 weeks on a 28day cycle was defined as the maximum tolerated dose.

| DISCUSSION
This open-label single-institution phase I trial confirmed the safety and tolerability of sapanisertib in combination with ziv-aflibercept in heavily pre-treated patients with advanced refractory cancer.The safety profile of mTORC1/2 inhibitor sapanisertib in combination with ziv-aflibercept was generally tolerable, and toxicities observed with both agents were mostly grade 1-2 and consistent with previously published attributions in the literature.Sapanisertib 4 mg orally 3 days on and 4 days off plus 3 mg/kg zivaflibercept IV every 2 weeks on a 28-day cycle was defined as the maximum tolerated dose and recommended phase II dose.No new safety signals were identified for either agent.
Recently, a phase I study of sapanisertib, a dual mTORC1/2 inhibitor, was conducted in patients with advanced solid tumors with expansion cohorts in renal, endometrial, or bladder cancer. 22Here, Voss et al. confirmed the pharmacodynamic effect of sapanisertib on downstream effectors of TORC1 (p4EBP1 and pS6) and TORC2 (pPRAS40 and pNDRG1), with treatment-related decreases in p4EBP1, pS6, pPRAS40, and pNDRG1 using single agent sapanisertib doses of ≥4 mg. 22Other groups have previously demonstrated treatment-related reductions in mTORC biomarkers, including TORC1/2 skin biomarkers (phosphorylated S6, 4EBP1, and PRAS40), 34 which support the dual TORC1/2 inhibitory activity of sapanisertib.In another study published this year, Paik et al demonstrated potent activity of TAK-228 in NSCLC models harboring Nrf2 activating alterations 35 and confirmed single agent clinical activity in genomically selected squamous cell lung cancer with NFE2L2/KEAP1 alterations, while also suggesting a role for combination therapy in treatment resistant patients. 35e demonstrate preliminary anti-tumor activity using sapanisertib in combination with ziv-aflibercept across tumor types.While the overall response rate of the combination was muted (4%), most patients derived clinical benefit, with 80% of evaluable patients achieving disease control (SD and/or PR); 37 patients (74%) achieved stable disease.Of the patients with stable disease as best response, four of these patients had initial unconfirmed partial responses.The two patients with unconfirmed PRs on imaging had AKT E17K hotspot mutations, while of the two patients who achieved confirmed partial responses, one patient had dual TSC1 mutations, suggesting a potential role for this combination in patients with activation of the mTOR/AKT/PI3K pathway.
There are several limitations of this study.The duration of the study was prolonged due to delayed enrollment because of drug formulation changes and dosing schedule modification.The inclusion of many patients with co-mutations in addition to mTOR/AKT/PI3K pathway alterations also may have confounded the interpretation of overall response to the combination strategy.
In conclusion, we demonstrate that the combination strategy utilizing sapanisertib and ziv-aflibercept is safe and well tolerated, with some clinical benefit demonstrated in our cohort of heavily pre-treated patients with and without mTOR/AKT/PI3K pathway alterations.Further study of sapanisertib in combination with  ziv-aflibercept may be warranted at the recommended phase II dose of sapanisertib 4 mg orally, 3 days on and 4 days off, plus 3 mg/kg ziv-aflibercept IV every 2 weeks on a 28-day cycle.In conclusion, sapanisertib in combination with ziv-aflibercept has exhibited preliminary anti-tumor activity and a favorable safety profile in late-line patients with advanced solid tumors.

1
Waterfall plot showing best overall response of evaluable patients on trial.Among 55 patients in the data set, 50 patients were response evaluable, of whom 5 had non-measurable disease by RECIST v1.1 criteria.Two patients (4%) achieved confirmed partial response (PR), 37 had stable disease (SD) (74%) (including four patients with unconfirmed partial responses); 11 patients (22%) had progression of disease (PD) as best response.Five patients were NE for response.The five patients with non-measurable, but evaluable disease and are denoted by *; two of these patients had clinical PD, and three of these patients achieved stable disease.F I G U R E 2Waterfall plot showing best overall response of evaluable patients in the dose expansion cohort.Ten patients were treated in the dose expansion cohort; among nine evaluable patients, one patient achieved confirmed partial response, five patients had stable disease as best response and three patients had progression of disease (one with clinical progression of disease), arbitrarily assigned 20% increase from baseline measurements).*Patient with Li Fraumeni syndrome.

Note: 3
Most common treated-related adverse events are summarized by severity, severity based on NCI CTCAE criteria version 5. Summary of dose escalations and dose-limiting toxicities in patients treated on study.
Baseline patient demographics.